Type 2 diabetes mellitus (T2DM) is characterized by defects in insulin action and insulin secretion. Disturbances in free fatty acid (FFA) metabolism also are a characteristic feature of T2DM and are observed in genetically predisposed individuals before the onset of overt diabetes. This raises the interesting possibility that FFA act as metabolic messengers which, when released in increased amounts, impair insulin action in insulin target tissues, i.e., "lipotoxicity". Much evidence also indicates that tissue fat content is increased in T2DM. We hypothesize that tissue lipid overload decreases expression of PGC-1, NRF-1, and multiple mitochondrial genes involved in oxidative phosphorylation. The resultant impairment in mitochondrial function leads to impaired substrate oxidation and accumulation of toxic lipid metabolites that inhibit insulin signaling and cause insulin resistance. In vivo and in vitro studies also suggest that increased hexosamine flux inhibits expression of PGC-1 and multiple mitochondrial genes involved in oxidative phosphorylation, i.e. "glucotoxicity". In the presence of hyperglycemia, an increase in malonyl CoA would be expected to further impair muscle fat and glucose oxidation by inhibiting CPTI, leading to an increase in toxic intracellular lipid metabolites and worsening of the insulin resistance, i.e. "glucolipotoxicity". In the present grant we shall examine the mechanisms of FFA-induced and hyperglycemia-induced insulin resistance. Using the insulin clamp with vastus lateralis muscle biopsy, magnetic resonance spectroscopy, and in vivo and in vitro evaluation of mitochondrial function, we shall examine the effect of elevated plasma FFA alone, increased glucosamine (glucose) alone, and the combination of elevated plasma glucosamine (glucose) plus elevated plasma FFA on whole body (muscle) insulin-stimulated glucose disposal/glucose oxidation/glycogen synthesis, insulin signaling, and mitochondrial gene expression and function in healthy NGT-insulin sensitive subjects. We also will examine the effect of acipimox (a potent inhibitor of lipolysis) and the effect of a highly specific inhibitor of renal tubular (SGLT2) glucose transport (BMS 512148) on the preceding parameters in T2DM subjects. These treatments reduce plasma FFA/deplete lipid from muscle and reduce plasma glucose levels, respectively. Therefore, we hypothesize that these interventions will increase PGC-1/NRF- 1/mitochondrial gene expression, improve mitochondrial function, and enhance insulin sensitivity/secretion. Lastly, we will examine the effect of combined acipimox/BMS 512148 therapy on the above paramters in T2DM. We believe that these studies will yield new insights into the etiology of insulin resistance in T2DM and identify novel therapeutic approaches to reverse the defects in insulin action and restore normoglycemia.